Knowledge of genetic information in the form of the nucleotide sequence of genes is critical to an understanding of various biological phenomenon such as cell development and differentiation, organism growth and reproduction, the underlying causes of disease, etc. For example, proteins serve a variety of structural and catalytic functions. These properties of proteins, however, are a function of the amino acid sequence of the protein, which in turn is encoded by nucleic acid sequences. Nucleic acids can also play a more direct role in cellular processes by functioning in the control and regulation of gene expression.
A variety of hybridization techniques have been developed to conduct various types of nucleic acid analyses to gain insight into how genetic information functions in these different types of biological processes. Typically, hybridization techniques involve the binding of certain target nucleic acids by nucleic acid probes under controlled conditions such that hybridization only occurs between complementary sequences. Using such hybridization techniques, it is possible to conduct gene expression studies, sequence checking studies and determine the sequence of nucleic acids of unknown sequence, as well as a variety of other types of analysis.
Gene expression studies are important because differential expression of genes has been shown to be associated with cell development, cell differentiation, disease states and adaptation to various environmental stimuli. For example, many diseases have been characterized by differences in the expression levels of various genes either through change in copy number of the genetic DNA or through alterations in levels of transcription. In certain diseases, infection is frequently characterized by elevated expression of genes from a particular virus.
Sequence checking refers to methods in which samples containing nucleic acid targets are analyzed to detect the presence of a sequence of interest. This type of analysis has utility in diverse applications, including research, clinical diagnostics, quality control, etc. One particular type of sequence checking which is particularly important is the identification of polymorphisms, which are variations in the genetic code. Often polymorphisms take the form of a change in a single nucleotide and are called single nucleotide polymorphisms (SNPs). In other instances, the polymorphism may exist as a stretch of repeating sequences that vary in length among different individuals. In those instances in which these variations exist in a significant percentage of the population, they can readily be used as markers linked to genes involved in mono- and polygenic traits. Thus, analysis of polymorphisms can play an important role in locating, identifying and characterizing genes which are responsible for specific traits. In particular, polymorphisms can be used to identify genes responsible for certain diseases. Similarly, diagnostic tests can also be developed to detect polymorphisms known to be associated with certain diseases or disorders.
Hybridization techniques can also successfully be used in sequencing nucleic acids of unknown sequence. Such methods typically are considerably faster than conventional sequencing techniques.
Chips to which nucleic acid probes are attached can be used to conduct nucleic acid analyses. Probes can be attached at specific locations on the chip; these locations are often referred to as elements or sites. In some applications, the chip may include many elements arranged in the form of an array. Genetic methods utilizing arrays on chips have the advantage of allowing for parallel processing that can dramatically increase the rate at which analyzes can be conducted as compared to conventional methods which often require laborious electrophoretic separations. However, the current nucleic acid methods using chips typically require complex labeling procedures in order to identify which nucleic acid probes have hybridized with a target molecule. Moreover, the methods frequently involve complicated stringency washes in order to minimize binding between probes and targets which are not fully complementary.
The present invention provides new methods for conducting various types of nucleic acid analysis in which hybridization of probe and target sequences can be detected directly, thereby allowing the analyses to be simplified relative to existing methodologies.